Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica

Abstract

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

Dual CRISPR‐Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica

“…The oleaginous yeast Yarrowia lipolytica has been explored as a platform for the production of lipid‐based fuels and chemicals . Strain engineering has been aided by recent developments in CRISPR‐Cas9 technology that provide powerful tools for targeted genome editing in Y. lipolytica .…”

A Strong Hybrid Fatty Acid Inducible Transcriptional Sensor Built From Yarrowia lipolytica Upstream Activating and Regulatory Sequences

“…Y. lipolytica strain PO1f (ATCC MYA‐2613; MATa leu2−270 ura3−302 xpr2−322 axp ) was purchased from ATCC. Y. lipolytica transformations were done using the lithium acetate method as previously described with a minor modification for cell propagation after the transformation . Briefly, following the heat shock step, the cells were mixed with 800 μL of 0.1 M lithium acetate, spun down at 6000 × g for 2 min (4 °C) and re‐suspended in 100 μL of 0.1 M LiAc buffer prior to inoculating 2 mL cultures that were grown in 14 mL culture tubes.…”

“…Similarly, it has been quite simple to identify loss‐of‐strength mutations in promoters . Rational approaches to increasing promoter strength have focused on hybrid promoters, where heuristically identified, well‐defined DNA elements called upstream activating sequences (UASs) are placed in front of a core promoter sequence and can be used in a modular fashion to tune transcription strength . While improving the strength of yeast promoters has been successful, the toolkit is currently lacking many options for inducible promoters.…”

“…In recent years, there has been an increase in genetic tools for metabolic engineering in the oleaginous yeast, Y. lipolytica . The advancement of genetic tools has enabled metabolic engineering in this microorganism to produce a significant amount of FAs and lipids from different substrates . Given the relative weakness of endogenous Y. lipolytica promoters, hybrid promoters have had great success in heterologous gene expression .…”

“…Strong expression of genes is enabled by hybrid promoters; however, the current set of strong hybrid promoters is limited. Although tunable in expression strength, hybrid promoters sometimes elicit carbon source dependent regulation from the UAS, as was previously demonstrated using the POX2 UAS1B . While this property might be used to create regulated promoters, the inducibility is neither rational nor tightly regulated.…”